Standardized Bovine Blood Analysis

Blood samples were collected from all cows all at one time. The samples were collected in the morning, before the feed distribution, by venipuncture from the jugular vein, using 10-mL Li-heparin treated tubes (Vacuette, containing 18 IU of Li-heparin mL⁻¹, Kremsmünster, Austria). Samples were immediately cooled in an ice-water bath after collection.
The blood was centrifuged (3500 × g for 16 min at 4 °C) and the plasma obtained was separated into two aliquots: the first fraction was immediately used to collect the infrared spectra; the second one was stocked at −20 °C until metabolites analysis and the results were used as calibration values.
Plasma metabolites used as calibrating values were analyzed at 37 °C by an automated clinical analyzer (ILAB 600, Instrumentation Laboratory, Lexington, MA), using the methodology showed in Table 1. Commercial kits were used to measure glucose, total cholesterol, urea, calcium, inorganic phosphorus, magnesium, total protein, albumin, total bilirubin, and creatinine (Instrumentation Laboratory SpA, Werfen, Monza, Milan, Italy), NEFA and zinc (Wako, Chemicals GmbH, Neuss, Germany), and β-OH-butyric acid (BHBA, kit Ranbut, Randox Laboratories Limited, Crumlin, County Antrim, United Kingdom Randox, UK). A Trinder end point [Glucose oxidase (GOD)/Peroxidase (POD)] was used to measure glucose. Total cholesterol (cholesterol and cholesterol esters) was also measured using Trinder end point [Cholesterol oxidase (CHOD)/Peroxidase (POD)], after a hydrolysis of cholesterol esters to free cholesterol. Urea was measured with end point method using the couple urease glutamate dehydrogenase (GLDH) enzyme system. Colorimetric methodology based on the reaction of calcium with o-cresolphthalein complexone was used to measure Ca with end point method. Inorganic phosphorus was measured with end point UV method, based on the reaction between phosphate ions in an acidic medium with ammonium molybdate to form a phosphomolybdate complex. The magnesium determination was based on the reaction of magnesium with Xylidyl Blue (as chelator) at alkaline pH, which yields a purple colored complex. Total protein were measured with the modified biuret methodology, based on the reaction of peptide bonds with Cu⁺⁺ ions in alkaline solution to form a colored complex. Albumin was measured with an end point colorimetric method, based on the binding between albumin with green bromocresol resulting in a spectral change of the dye from yellow to green. Total bilirubin was determined with an end point analysis using modified Jendrassik-Grof method, based on the reaction between total bilirubin with diazotized sulfanilic acid in presence of lithium dodecylsulfate to form azobilirubin. Creatinine was analyzed with an end point colorimetric method, based on the reaction of creatinine with picric acid under alkaline conditions. NEFA were determined with an Trinder end point [Acyl coenzyme A oxidase (ACOD)/Peroxidase (POD)] assay, after the acylation of coenzyme A by NEFA contained in the sample. BHBA was measured with a kinetic UV method, based on the oxidation od D-3 hydroxybutyrate to acetoacetate by 3-Hydroxybutyrate dehydrogenase. Electrolytes, Na, K, and Cl, were measured using a potentiometric system, with specific electrodes. Kinetic analysis was adopted to determine the activity of enzymes: alkaline phosphatase (AP, EC 3.1.3.1), aspartate aminotransferase (AST, EC 2.6.1.1), γ-glutamyltransferase (GGT, EC 2.3.2.2) using Instrumentation Laboratory kits (Instrumentation Laboratory SpA, Werfen, Monza, Milan, Italy). Ceruloplasmin was measured using the method described by Sunderman and Nomoto [16 (link)], adapted to ILAB 600 condition. The method is based on measurement of p-phenylenediamine dihydrochloride oxidation by the oxidase activity of ceruloplasmin. Finally, haptoglobin was measured using the method described by Skinner et al. [17 (link)] and Owen et al. [18 (link)] and adapted to ILAB 600 condition. The method is based on peroxidase activity of methaemoglobin-haptoglobin complex measured by the rate of oxidation of guaiacol (hydrogen donor) in presence of hydrogen peroxide (oxidizing substrate).Table 1

Methodologies used to measure the plasma parameters with reference methods

Parameter	Methodology	Wavelength (nm)	CVa
Glucose	Endpoint	510	1.50
Total cholesterol	Endpoint	510	2.10
NEFAb	Endpoint	546	1.50
BHBAc	Endpoint	340	5.25
Urea	Endpoint	340	1.20
Creatinine	Endpoint	510	5.40
Ca	Endpoint	570	1.40
Inorganig P	Endpoint	340	2.00
Mg	Rate	340	1.40
Na	ISE deviceg		0.90
K	ISE deviceg		1.30
Cl	ISE deviceg		1.50
Zn	Endpoint	546
Ceruloplasmin	Endpoint	546	3.48
Total protein	Endpoint	546	1.20
Albumin	Endpoint	600	1.80
Total bilirubin	Endpoint	546	6.70
Haptoglobin	Endpoint	450	13.54
ASTd	Rate	340	2.10
GGTe	Rate	405	3.72
APf	Rate	405	1.70

^aCalculared on the results obtained between runs according to the National Committee for Clinical Laboratory Standards (Document EP3-T: Guidelines for Manufacturers for Establishing

Performance Claims for Clinical Chemistry Methods, Replication Experiment

Evaluation”, Villanova, PA, 1982.)

^bNon esterified fatty acids;

^cβ-OH-butyric acid;

^dAspartate amino transferase

^eγ-glutamyl transferase

^fAlkaline phosphatase

^gIon selective electrodes